Our current studies focus on protein targeting to and translocation across the E. coli IM. Most of the major factors that promote the targeting and translocation reactions are extremely highly conserved, and E. coli has proven to be an outstanding model organism in which to study these conserved factors. In addition, obtaining a better understanding of protein translocation across the IM is of great interest to the biotechnology industry, which has sought to optimize the production of secreted proteins in E. coli. Finally, it may ultimately be possible to exploit small differences in the protein targeting and translocation machinery in bacteria and eukaryotic cells to develop novel antimicrobials. Consistent with the work of other laboratories, we found several years ago that most presecretory proteins are bypassed by SRP and targeted to the IM posttranslationally by molecular chaperones such as SecB. Chaperones are required to prevent fully synthesized proteins from folding into a compact, translocation-incompetent conformation. More recently we have been trying to understand the basis of targeting pathway selection. We found that bacterial SRP has a more restricted substrate specificity than its mammalian counterpart and recognizes targeting signals based primarily on their hydrophobicity. Bacterial SRP tends to recognize the membrane-spanning segments of integral membrane proteins (which serve as internal targeting signals) much more often than the cleaved signal peptides of presecretory proteins largely because they are more hydrophobic. Substrate hydrophobicity is not the only factor that dictates targeting pathway selection, however, and we have found that the presence of basic residues near the N-terminus of signal peptides can influence SRP recognition. In very recent studies we have also found that cotranslational targeting by the SRP pathway is necessary, but not sufficient to guarantee the export of proteins that fold very rapidly.